Preparation Method for Charge Reversaland Reversibly Crosslinked Redox-Sensitive Nanomicelles

ABSTRACT

Disclosed is a preparation method for charge reversal and reversibly crosslinked redox-sensitive nanomicelles, falling within the technical field of biomedical materials. The method comprises: synthesizing thiocinamide from lipoic acid and ethylenediamine under an N,N′-carbonyl diimidazole catalyst; and polymerizing thiocinamide, polyethylene glycol diglycidyl ether and lysine through a nucleophilic addition mechanism to prepare a poly(lysine-co-polyethylene glycol diglycidyl ether-co-thiocinamide) terpolymer. The micelle is endowed with excellent anti-protein nonspecific adsorption and enhanced cell uptake property through a self-assembly and protonation/deprotonation action; and a disulfide bond in lipoyl may form a linear polydisulfide structure under the action of 1,4-dithiothreitol, so that a micelle core is crosslinked, and a crosslinked structure is destroyed in the cell under a redox condition, and controlled release of a drug can be achieved. The Nanomicelle of the present invention is expected to be a carrier of drugs for treating cancers.

TECHNICAL FIELD

The present invention relates to a preparation method for chargereversal and reversibly crosslinked redox-sensitive nanomicelles,falling within the technical field of biomedical materials.

BACKGROUND

After entering a human body, a drug nano-carrier is diluted by a greatamount of body fluid, and thus becomes less stable. In order to increasestability, the carrier is subjected to hydrophilic shell crosslinking,hydrophobic core crosslinking or core-shell interface crosslinking, soas to reduce carrier dissociation caused by dilution. However, thesetraditional crosslinking manners also reduce the efficiency of drugrelease while stabilizing the carrier. Furthermore, a very small numberof crosslinking structures are biocompatible and biodegradable. Thisgreatly limits application of these carriers in the field of biologicalmedicine.

Complex protein components in blood easily react with carriers carryingpositive charges or active groups in the carriers to make the carriersgathered and removed out of the body. Carriers smoothly enteringtargeted parts reduce uptake of drug carriers by cells due to cellmembrane rejection, thereby influencing the bioavailability of drugs.Recently, researchers apply a charge transfer caused by acid-sensitivebond breakage to drug carriers, such that the carriers are negativelycharged during body circulation, thereby effectively avoidinginteraction with proteins in blood; and when entering tumor tissues, thecarriers are positively charged in a weak acid environment, so thatinteraction between the carriers and cancer cells can be enhanced,thereby improving uptake of carriers by cells. The charge transfer isadvantageous in endowing the carriers with anti-protein adsorptionperformance and increasing cell uptake capability.

At present, there are few reports about reversible core-crosslinked drugcarriers with reversible charges, reactions needed for providing chargereversal properties and crosslinking structures are complex, and most offormed materials cannot meet biodegradability, thereby reducing theapplication feasibility of such materials.

SUMMARY

To solve the foregoing technical problem, the objective of the presentinvention is to provide a preparation method for a reversiblycrosslinked redox-sensitive Nanomicelle with reversible charges andexcellent biocompatibility.

The present invention provides a preparation method for a reversiblycrosslinked redox-sensitive Nanomicelle with reversible charges. Thepolymer s synthesized from three monomers namely polyethylene glycoldiglycidyl ether, lysine and thiocinamide through a nucleophilicring-opening reaction. A synthesis diagram is as shown in FIG. 1. Toform a micelle in an appropriate hydrophilic-hydrophobic ratio, themolecular weight of the adopted polyethylene glycol diglycidyl ether is315 g/mol. The feeding mole ratio of the three monomers is in a range of1:0.9:0.1 to 1:0.1:0.9, and a component ratio can be adjusted to controlthe forming of the micelle and isoelectric points thereof. Three monomerunits have respective functions in a Nanomicelle as follows. 1)Polyethylene glycol diglycidyl ether serves as a bridge connecting twosmall functional molecules and is excellent in biocompatibility. 2)Lysine is one of amino acids essential to a human body, carboxyl andamino in molecules of lysine can achieve protonation/deprotonation underdifferent pH conditions so as to control charges on the surface of acarrier, and meanwhile, blood anti-protein adsorption and enhanced celluptake property are achieved. 3) Lipoic acid is an antioxidant in thehuman body, a disulfide bond in a five-membered ring thereof can form acrosslinking structure with a plurality of adjacent disulfide bondsunder the action of a catalytic amount of DTT, the crosslinkingstructure can be broken under the action of a high-concentration redox(such as GSH), and the special reversible crosslinking structure canstabilize body circulation of the carrier and can be destroyed under theaction of high-concentration GSH inside a cell, thereby enhancing thetargeted release capability of a drug.

In an embodiment of the present invention, the mole ratio ofpolyethylene glycol diglycidyl ether, lysine and thiocinamide is1:0.7:0.3.

In an embodiment of the present invention, the mole ratio ofpolyethylene glycol diglycidyl ether, lysine and thiocinamide is 1:1:0.

In an embodiment of the present invention, the mole ratio ofpolyethylene glycol diglycidyl ether, lysine and thiocinamide is1:0.5:0.5.

In an embodiment of the present invention, a mole ratio of polyethyleneglycol diglycidyl ether, lysine and thiocinamide is 1:0.3:0.7.

The present invention also provides a preparation method for areversibly crosslinked redox-sensitive Nanomicelle with reversiblecharges, sequentially comprising the following steps:

1) generating Lipoyl ethylene diamine (LAE) through a reaction betweenlipoic acid and ethylenediamine under the action of a catalystN,N′-carbonyl diimidazole;

2) obtaining a coarse terpolymer solution from thiocinamide,polyethylene glycol diglycidyl ether with different molecular weights,and lysine through a nucleophilic addition reaction;

3) dialyzing and drying a coarse reaction solution to obtain purepolymer powder;

4) dissolving the powder, and dropwise adding ultra-pure water into theterpolymer solution slowly under continuous stirring;

5) after the step of dropwise adding the water is ended, stirring for aperiod of time, transferring the solution into a dialysis bag, andcarrying out dialysis treatment to obtain a non-crosslinked Nanomicelle;and

6) under a nitrogen flow, adding a catalytic amount of DTT solution intoa non-crosslinked micelle solution, carrying out stirring for 24 hours,and then carrying out dialysis for 24 hours, so as to obtain acrosslinked Nanomicelle solution.

Specifically, in the step 1), a catalyst adopted for an amidationreaction between lipoic acid and ethylenediamine is N,N′-carbonyldiimidazole, a reaction solvent is chloroform, and reaction products areextracted by using 10% sodium chloride and 1M sodium hydroxiderespectively.

Specifically, in the step 2), polyethylene glycol diglycidyl ether ofwhich the molecular weight is 315 g/mol is adopted for a nucleophilicreaction, and under this molecular weight, a polymerhydrophilic-hydrophobic ratio can be well controlled, so as to form amicelle.

Specifically, in the step 2), the mole ratio of three reactioncomponents is in a range of 1:0.9:0.1 to 1:0.1:0.9, and an optimumreaction ratio can be selected according to an appropriate particle sizeand isoelectric point. The reaction solvent is a mixed solution ofmethyl alcohol and water, the volume ratio is 1:1, and the action iscarried out for 3 days at the temperature of 50° C. under the nitrogenprotection.

Specifically, in the step 3), a dialysis bag of which the molecularweight cutoff is 3500 is adopted for dialysis, and dialysis fluid is amixed solution of methyl alcohol and water (v:v=1:1). The objective ofthe dialysis is to remove polymers with low molecular weight in favor offorming a micelle with a uniform particle size.

Specifically, in the step 4), a polymer is dissolved in dimethylsulfoxide, and after the polymer is fully dissolved, ultra-pure water isdropwise added at the speed of 30 s/drop.

Specifically, in the step 5), after the step of dropwise adding thewater is ended, stirring is carried out for two hours, the solution istransferred into the dialysis bag of which the molecular weight cutoffis 3500, and dialysis is carried out for not less than 24 hours, so asto obtain a pure non-crosslinked Nanomicelle solution.

Specifically, in the step 6), DTT is 10 mol % of lipoyl, and excessiveDTT may result in that a disulfide bond is thoroughly broken and cannotbe crosslinked. The reaction is carried out under a dark condition atthe temperature of 25° C.

The present invention also provides application of a reversiblycrosslinked redox-sensitive Nanomicelle with reversible charges inpreparation of chemotherapeutic drug carriers. A micelle carries ahydrophobic anti-cancer drug through hydrophobic interaction, andmeanwhile, a disulfide bond in a hydrophobic chain segment thiocinamidecan be crosslinked under the action of a catalytic amount of DTT to forma more stable drug-carrying Nanomicelle. Due to a special physiologicalenvironment in a cancer cell, the content of GSH in a cytoplasm and acell nucleus of the cancer cell can be up to 1000 times that in bodyblood, so that after a Nanomicelle which originally and stably entrapsdrug for circulation in the blood enters the cancer cell, thecrosslinked disulfide bond is destroyed by high-concentration GSH redox,thereby achieving controlled release of a drug in the cell.

By means of the foregoing solutions, the present invention at least hasthe advantages as follows.

1. The preparation method of the present invention synthesizes a polymerby utilizing nucleophilic addition without addition of a catalyst, thereaction being mild and efficient.

2. During the nucleophilic addition process of lysine, the possibilityof amino on a pendant group participating in the reaction under theinfluence of carboxyl is greatly reduced; and protonation/deprotonationof the remaining amino and carboxyl under different pH conditions canachieve excellent anti-protein adsorption property during bodycirculation.

3. The CMC value of the polymer Nanomicelle prepared in the presentinvention is in a range of 0.011 to 0.038 mg/mL, the Nanomicelle isnegatively charged under the pH value of 7.4, and the charges on thesurface of the micelle are transferred into positive charges under thepH value of 6.5, so that when the micelle reaches tumor tissues, thecharges on the surface of the micelle are transferred into positivecharges, uptake of drug carriers by cancer cells can be improved bymeans of an electrostatic action, and it is unnecessary to be bondedwith a specific ligand, thereby not increasing the reaction difficulty.

4. A disulfide bond in the Nanomicelle can form a core crosslinkedstable structure under the action of catalytic-amount DTT, so as toachieve stable circulation in a human body; and the structure is brokenin quick response in a cell nucleus and cytoplasm containinghigh-concentration GSH. The Nanomicelle can be used for carrying ananti-cancer drug and achieving targeted release of the drug.

5. A polymer synthesized from raw materials namely polyethylene glycoldiglycidyl ether and endogenous substances of a human body has excellentbiocompatibility, the cell survival rates of cells L929 and Hela inNanomicelle solutions with different concentrations being 90% or more.

The above is only the summary of the technical solutions of the presentinvention. In order to more clearly understand the technical means ofthe present invention and to implement in accordance with the content ofthe description, the detailed description will be made hereinafter inconjunction with preferred embodiments of the present invention and thedrawings.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a schematic diagram of forming a crosslinked micelle in thepresent invention;

FIG. 2 is transmission electron microscope images of a reversiblycrosslinked redox-sensitive Nanomicelle with reversible charges in thepresent invention before and after being crosslinked,

where N4 is representative of a reversibly crosslinked redox-sensitiveNanomicelle with reversible charges obtained by a reaction amongpolyethylene glycol diglycidyl ether, thiocinamide and lipoic acid in amole ratio of 1:0.3:0.7;

FIG. 3 is a critical micelle concentration of a reversibly crosslinkedredox-sensitive Nanomicelle with reversible charges in the presentinvention, where N1, N2, N3 and N4 are representatives of a reversiblycrosslinked redox-sensitive Nanomicelle with reversible charges obtainedby a reaction among polyethylene glycol diglycidyl ether, thiocinamideand lipoic acid in a mole ratio of 1:1:0, 1:0.7:0.3, 1:0.5:0.5 and1:0.3:0.7 respectively, and C is a polymer micelle concentration(mg/mL);

FIG. 4 is a zeta potential of a reversibly crosslinked redox-sensitiveNanomicelle with reversible charges in the present invention underdifferent pH values, where N1, N2, N3 and N4 are representatives of areversibly crosslinked redox-sensitive Nanomicelle with reversiblecharges obtained by a reaction among polyethylene glycol diglycidylether, thiocinamide and lipoic acid in a mole ratio of 1:1:0, 1:0.7:0.3,1:0.5:0.5 and 1:0.3:0.7 respectively;

FIG. 5 is a particle size change of a reversibly crosslinkedredox-sensitive Nanomicelle with reversible charges in the presentinvention after being incubated in a protein solution for 24 hours,where in a reversibly crosslinked redox-sensitive Nanomicelle withreversible charges obtained by a reaction among polyethylene glycoldiglycidyl ether, thiocinamide and lipoic acid in a mole ratio of1:0.3:0.7, BSA serves as a model protein of which the concentration is45 g/L;

FIG. 6 is a particle size change of a reversibly crosslinkedredox-sensitive Nanomicelle with reversible charges in the presentinvention, where N4 is representative of a reversibly crosslinkedredox-sensitive Nanomicelle with reversible charges obtained by areaction among polyethylene glycol diglycidyl ether, thiocinamide andlipoic acid in a mole ratio of 1:0.3:0.7;

FIG. 7 is a fluorescence intensity change of a reversibly crosslinkedredox-sensitive Nanomicelle with reversible charges in the presentinvention in a 10 mmol/L glutathione solution, where a reversiblycrosslinked redox-sensitive Nanomicelle with reversible charges isobtained by a reaction among polyethylene glycol diglycidyl ether,thiocinamide and lipoic acid in a mole ratio of 1:0.3:0.7;

FIG. 8 is a cytotoxicity result of a reversibly crosslinkedredox-sensitive Nanomicelle with reversible charges in the presentinvention.

DETAILED DESCRIPTION

The detailed description of the invention will be made below inconjunction with the drawings and the embodiments. The followingexamples are used to illustrate the present invention, but not to limitthe scope of the present invention.

EXAMPLE 1

1) Synthesis of thiocinamide:

3.00 g of lipoic acid (1.45×10−2 mol) and 2.59 g of N,N′-carbonyldiimidazole (1.60×10−2 mol) are weighed, and dissolved in 30 mL ofchloroform, and react for 1 hour at the temperature of 25° C. under thenitrogen protection, and then a mixed solution is transferred into adropping funnel and dropwise added into a uniformly-stirred chloroform(30 mL) solution of ethylenediamine (8 mL, 0.12 mol), and reacts for 12hours at the temperature of 25° C. under the nitrogen protection. Afterthe reaction is ended, a reaction mixed solution is transferred into aseparating funnel and extracted by using 10% NaCl (100 mL) and 1M NaOH(100 mL) respectively, organic phases are collected, and an organicsolvent is removed by rotary evaporation to obtain a yellow gelatinouscompound thiocinamide (2.2 g, 61%).

2) Synthesis of poly(lysine-co-polyethylene glycol diglycidylether-co-thiocinamide):

315 mg of polyethylene glycol diglycidyl ether (1 mmol), 102 mg oflysine (0.7 mmol) and 75 mg of thiocinamide (0.3 mmol) are weighedrespectively and dissolved in 3 mL of a mixed solution of methyl alcoholand water (V:V=1:1), and react for 72 hours in a 25 mL single-openingflask at the temperature of 50° C. under the nitrogen protection. Afterthe reaction is ended, a coarse solution is transferred into a dialysisbag of which the molecular weight cutoff is 3500, dialyzed for 3 days,and freeze-dried to obtain a yellow product. The correspondingdissolution ratio of polyethylene glycol diglycidyl ether, thiocinamideand lysine in a mixed solution is as shown in Table 1. The presentinvention provides only 4 terpolymer synthesis formulas. During aspecific using process, the ratio of three monomers is adjustedaccording to application requirements of a Nanomicelle for differentproperties.

TABLE 1 Table of terpolymer synthesis formula Polyethylene glycolPolyethylene diglycidylether:thiocinamide: glycol Nanomicelle lysinediglycidyl number (feeding mole ratio) ether/mg Thiocinamide/mgLysine/mg N₁ 1:1:0 315 248 0 N₂ 1:0.7:0.3 315 174 44 N₃ 1:0.5:0.5 315124 73 N₄ 1:0.3:0.7 315 75 102

3) Preparation of reversibly crosslinked redox-sensitive Nanomicellewith reversible charges:

10 mg of a polymer is weighed and dissolved in 1 mL of dimethylsulfoxide, and after the polymer is fully dissolved, 8 mL of ultra-purewater is dropwise added. After the step of dropwise adding the water isended, stirring is carried out for 2 hours, the solution is transferredinto the dialysis bag of which the molecular weight cutoff is 3500, anddialysis is carried out for 72 hours, so as to obtain a purenon-crosslinked Nanomicelle solution. The Nanomicelle is crosslinkedunder the atmosphere of feeding N₂, and a catalytic amount of DTT isadded. Specifically, a DTT (77 μg, 0.5 μmol) solution is added into 10mL of non-crosslinked N₄ micelle (1 mg/mL) solution, and stirred for 24hours under a dark condition at the temperature of 25° C. Dialysis iscarried out for 24 hours by means of a dialysis method to obtain a purecrosslinked Nanomicelle solution. FIG. 2 is transmission electronmicroscope images of a reversibly crosslinked redox-sensitiveNanomicelle with reversible charges before and after being crosslinked,where N₄ corresponds to a terpolymer obtained by a formula No. N₄ inTable 1, the micelle is a uniform and regular spherical micelle beforeand after being crosslinked, and the particle size of the crosslinkedmicelle is reduced by about 20 nm.

EXAMPLE 2

Measurement of reversibly crosslinked redox-sensitive Nanomicelle (CMC)with reversible charges:

The reversibly crosslinked redox-sensitive Nanomicelle solution withreversible charges obtained in the example 1 is diluted into a series ofmicelle solutions with different concentrations, and 4 mL of the micellesolution with each concentration is added into 30 μL of acetone solutionof pyrene of which the concentration is 1.622×10⁻⁵ g/mL, and incubatedin a constant-temperature shaking incubator under the atmosphere ofnitrogen for 24 hours at the temperature of 30° C. An emission spectrumis measured by utilizing a fluorescence spectrophotometer, afluorescence excitation wavelength A is set as 333 nm, a scanning rangeλ is in a range of 350 nm to 500 nm, both an excitation slit width andan emission slit width are 5 nm, and the thickness of a sample pool is 1cm. By measuring the fluorescence intensity of a series of Nanomicellesolutions with different concentrations at 373 nm (l₁) and 384 nm (l₃),a graph is drawn by taking a logarithm concentration as an X axis andl₁/l₃ as a Y axis, and the CMC value of a polymer Nanomicelle iscalculated by using a curve discontinuity point. From FIG. 3, it can beseen that N₁, N₂, N₃ and N₄ have lower CMC values namely 0.011, 0.020,0.030 and 0.038 mg/mL, which are increased along with the increase ofthe lysine content, and all have good anti-dilution stability.

EXAMPLE 3

pH sensitivity of reversibly crosslinked redox-sensitive Nanomicellewith reversible charges:

The reversibly crosslinked redox-sensitive Nanomicelle with reversiblecharges obtained in the example 1 is adjusted to different pH values byusing 0.1 mol/L sodium hydroxide solution and a hydrochloric acidsolution, and a Zeta potential is measured by utilizing a Zetapotentiometric analyzer. Consequently, from FIG. 4, it can be seen thatwhen carrier material components are not added with lysine (namely N₁),the Nanomicelle is hardly charged under the condition of pH 7.4; and asthe pH is decreased, positive charges on the surface of the Nanomicelleare increased. After the lysine component is added, carboxyl can bedeprotonated under an alkaline condition, so that the Nanomicelle isnegatively charged; the isoelectric point of a carrier can be adjustedby adjusting the ratio of three components; and when the mole ratio ofpolyethylene glycol diglycidyl ether, thiocinamide and lysine is1:0.3:0.7 (namely N₄), the isoelectric point thereof is 7.04, theNanomicelle can be negatively charged under the condition of pH 7.4, andcharges on the surface of the micelle are transferred into positivecharges under the condition of pH 6.5.

When the mole ratio of polyethylene glycol diglycidyl ether,thiocinamide and lysine is 1:0.3:0.7, the particle size of theNanomicelle is relatively appropriate, and when pH is equal to 7.04,charge transfer is achieved. The micelle can be endowed withanti-protein adsorption and enhanced cell uptake property. Therefore,Nanomicelles mentioned in the following examples of the presentinvention refer to the Nanomicelle under this ratio unless otherwisespecified.

EXAMPLE 4

Anti-protein nonspecific adsorption property of reversibly crosslinkedredox-sensitive Nanomicelle with reversible charges:

The reversibly crosslinked redox-sensitive Nanomicelle (N₄) withreversible charges obtained in the example 1 is put into a bovine serumalbumin solution of which the concentration is 45 g/L, a particle sizechange of the Nanomicelle is tested by using a laser light scatteringinstrument within different periods, and the protein adsorptioninfluence is observed. Consequently, as shown in FIG. 5, BSA in thefigure is bovine serum albumin, the particle size of the Nanomicelle N₄is not greatly changed within an observation period of 24 hours, and itis shown that a negatively-charged Nanomicelle can effectively rejectprotein adsorption and can be stably circulated in a human body.

EXAMPLE 5

Anti-dilution stability of a reversibly crosslinked redox-sensitiveNanomicelle with reversible charges:

The reversibly crosslinked redox-sensitive Nanomicelle (N₄, 0.5 mg/mL)with reversible charges obtained in the example 1 is diluted by 100times by using ultra-pure water to make its concentration lower than theCMC value, and the particle size change of the Nanomicelle is tested byusing a laser light scattering instrument. Consequently, as shown inFIG. 6, the particle size of the crosslinked Nanomicelle is reduced from187 nm to 162 nm, which is consistent with a result obtained by atransmission electron microscope image. The crosslinked Nanomicelle isdiluted to be the CMC value or below, it is discovered that the particlesize and the distribution thereof are not greatly changed, and it isshown that the crosslinked Nanomicelle has strong anti-dilutionstability and can be prevented from dissociation in case of dilution ofa great amount of body fluid.

EXAMPLE 6

Redox sensitivity of reversibly crosslinked redox-sensitive Nanomicellewith reversible charges:

The reversibly crosslinked redox-sensitive Nanomicelle (N₄, 4 mL, 1mg/mL) with reversible charges obtained in the example 1 is added intoan acetone solution of pyrene, so that the concentration of the pyre is5.0×10⁻⁶ mol/L.; the Nanomicelle is incubated in a constant-temperatureshaking incubator for 24 hours at the temperature of 30° C., and a GSHsolution is added to make the GSH concentration reach 10 mol/L; and thenwithin a specific time interval, an emission spectrum of the pyrene inthe micelle solution is measured by utilizing a fluorescencespectrophotometer, an emission wavelength is 395 nm, and both anexcitation slit width and an emission slit width are 5 nm. Consequently,as shown in FIG. 7, as time increases, the fluorescence intensity of thepyrene is gradually reduced, and it is shown that the pyrene enters ahydrophilic environment from a hydrophobic environment. The reason forthis phenomenon is that a disulfide bond of a micelle core is brokenunder the action of GSH to destroy an original micelle structure so asto release the pyrene wrapped by the micelle.

EXAMPLE 7

Biocompatibility of reversibly crosslinked redox-sensitive Nanomicellewith reversible charges:

A reversibly crosslinked redox-sensitive Nanomicelle N₄ with reversiblecharges obtained in the example 1 is taken as a research object. Thecytotoxicity of a Nanomicelle is tested by using an MTT method. Acervical cancer cell (Hela cell) or mouse fibroblast (L929 cell) growingin a logarithmic phase is digested by trypsin (0.25%), blown andscattered by using a DMEM culture medium containing 10% fetal calfserum, diluted into a cell suspension of which the concentration is1.0×10⁴ cells/mL by using the culture medium, inoculated to a 96-poreplate in 100 μL per pore, cultured in a constant-temperature incubatorfor 24 h, and then added into 100 μL of DMEM complete medium containingdifferent polymer concentrations (5, 25, 50, 100, 250 and 500 μg/mL)respectively after leaving from the culture medium. Each group isprovided with six complex pores, culture is carried out for 24 hours,and then the culture medium is removed. Each pore is added with 20 μL ofMTT solution (5 mg/mL), culture is continuously carried out for 4 hours,and then the solution in the pore plate is removed. Each pore is addedwith 150 μL of dimethyl sulfoxide, formazan generated from living cellsis dissolved by shaking, absorbancy is measured by using an ELIASA at570 nm, and the cell survival rate is calculated. Consequently, as shownin FIG. 8, the cell survival rates of cells L929 and Hela in Nanomicellesolutions with different concentrations are 90% or more, and it is shownthat the Nanomicelle has excellent biocompatibility.

1. A preparation method for charge reversal and reversibly crosslinkedredox-sensitive nanomicelles, sequentially comprising the followingsteps: 1) generating Lipoyl ethylene diamine (LAE) through a reactionbetween lipoic acid and ethylenediamine under the action of a catalyst;2) obtaining a coarse terpolymer solution from LAE, polyethylene glycoldiglycidyl ether and lysine through a nucleophilic addition reaction; 3)dialyzing a coarse reaction solution and drying to obtain a powder ofpure terpolymer; 4) dissolving the powder, and dropwise addingultra-pure water into the terpolymer solution slowly under continuousstirring; 5) after the step of dropwise adding the water is ended,stirring for a period of time, transferring the solution into a dialysisbag, and carrying out dialysis treatment to obtain a non-crosslinkednano-micelle; and 6) under a nitrogen flow, adding a catalytic amount of1,4-dithiothreitol solution (DTT) into a non-crosslinked micellesolution, carrying out stirring for 24 hours, and then carrying outdialysis for 24 hours, so as to obtain a crosslinked nano-micellesolution.
 2. The method according to claim 1, wherein in step 1), areaction solvent is chloroform, a reaction catalyst is N,N′-carbonyldiimidazole, the dosage is 80 to 90 Wt % of lipoic acid, and reactionproducts are extracted by using 10% sodium chloride and 1M sodiumhydroxide respectively.
 3. The method according to claim 1, wherein instep 2), the mole ratio of three components synthesizing a terpolymer isin a range of 1:0.9:0.1 to 1:0.1:0.9.
 4. The method according to claim1, wherein in step 2), a solvent adopted for a nucleophilic ring-openingreaction is a mixed solution of methyl alcohol and water, the volumeratio is 1:1, and the reaction is carried out for 3 days at atemperature of 50° C.
 5. The method according to claim 1, wherein instep 3), the molecular weight cutoff of the dialysis bag adopted fordialysis is 3500, dialysis fluid is a mixed solution of methyl alcoholand water, and the volume ratio is 1:1.
 6. The method according to claim1, wherein in step 4), a terpolymer is dissolved in dimethyl sulfoxide,and the dripping speed of the ultra-pure water is 30 s/drop.
 7. Themethod according to claim 1, wherein in step 5), after the step ofdropwise adding the water is ended, stirring is carried out for twohours, the solution is transferred into the dialysis bag of which themolecular weight cutoff is 3500, and dialysis treatment is carried outfor not less than 24 hours, so as to obtain a nano-micelle solution. 8.The method according to claim 1, wherein in step 6), 1,4-dithiothreitolis 10 mol % of lipoyl, and the reaction is carried out under a darkcondition at the temperature of 25° C.
 9. A redox-sensitive reversiblecrosslinked nano-micelle with reversible charges, prepared by using themethod of claim
 1. 10. A pharmaceutical composition comprising theredox-sensitive reversible crosslinked nano-micelle with reversiblecharges of claim 9.